title: The fumarate/succinate antiporter DcuB of Escherichia coli is a bifunctional protein with sites for regulation of DcuS-dependent gene expression.
abstract: DcuB of Escherichia coli catalyzes C4-dicarboxylate/succinate antiport during growth by fumarate respiration. The expression of genes of fumarate respiration, including the genes for DcuB (dcuB) and fumarate reductase (frdABCD) is transcriptionally activated by C4-dicarboxylates via the DcuS-DcuR two-component system, comprising the sensor kinase DcuS, which contains a periplasmic sensing domain for C4-dicarboxylates. Deletion or inactivation of dcuB caused constitutive expression of DcuS-regulated genes in the absence of C4-dicarboxylates. The effect was specific for DcuB and not observed after inactivation of the homologous DcuA or the more distantly related DcuC transporter. Random and site-directed mutation identified three point mutations (T394I, D398N, and K353A) in DcuB that caused a similar derepression as dcuB deletion, whereas the transport activity of the DcuB mutants was retained. Constitutive expression in the dcuB mutants depended on the presence of a functional DcuS-DcuR two-component system. Mutation of residues E79A, R83A, and R127A of DcuB, on the other hand, inactivated growth by fumarate respiration and transport of [14C]succinate, whereas the expression of dcuB'-'lacZ was not affected. Therefore, the antiporter DcuB is a bifunctional protein and has a regulatory function that is independent from transport, and sites for transport and regulation can be differentiated.
authors.affiliation: Institut fu¨r Mikrobiologie und Weinforschung, University of Mainz, Becherweg 15, 55099 Mainz, Germany.
authors.forename: Alexandra
authors.initials: A
authors.lastname: Kleefeld
authors.affiliation: Institut fu¨r Mikrobiologie und Weinforschung, University of Mainz, Becherweg 15, 55099 Mainz, Germany.
authors.forename: Bianca
authors.initials: B
authors.lastname: Ackermann
authors.affiliation: Institut fu¨r Mikrobiologie und Weinforschung, University of Mainz, Becherweg 15, 55099 Mainz, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Bauer
authors.affiliation: Institut fu¨r Mikrobiologie und Weinforschung, University of Mainz, Becherweg 15, 55099 Mainz, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Kra Mer
authors.affiliation: Institut fu¨r Mikrobiologie und Weinforschung, University of Mainz, Becherweg 15, 55099 Mainz, Germany. Electronic address: unden@uni-mainz.de.
authors.forename: Gottfried
authors.initials: G
authors.lastname: Unden
mesh_terms: D019943:Amino Acid Substitution; D017920:Antiporters / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D029382:Dicarboxylic Acid Transporters / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005650:Fumarates / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D055785:Gene Knockdown Techniques; D017136:Ion Transport / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D013385:Succinate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D013386:Succinates / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D017920:Antiporters; D004268:DNA-Binding Proteins; C115005:DcuR protein, E coli; D029382:Dicarboxylic Acid Transporters; D029968:Escherichia coli Proteins; D005650:Fumarates; D013386:Succinates; D014157:Transcription Factors; C086256:dcuB protein, E coli; C033727:fumarate reductase complex; D013385:Succinate Dehydrogenase; D011494:Protein Kinases; C469359:DcuS protein, E coli